Comparison of Proteomic Assessment Methods in Multiple Cohort Studies.
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ABSTRACT:
SUBMITTER: Raffield LM
PROVIDER: S-EPMC7425176 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Proteomics has been recently introduced in aquaculture research, and more methodological studies are needed to improve the quality of proteomics studies. Therefore, this work aims to compare three sample preparation methods for shotgun LC-MS/MS proteomics using tissues of two aquaculture species: liver of turbot Scophthalmus maximus and hepatopancreas of Mediterranean mussel Mytilus galloprovincialis. We compared the three most common sample preparation workflows for shotgun analysis: filter-aided sample preparation (FASP), suspension-trapping (S-Trap), and solid-phase-enhanced sample preparations (SP3). FASP showed the highest number of protein identifications for turbot samples, and S-Trap outperformed other methods for mussel samples. Subsequent functional analysis revealed a large number of Gene Ontology (GO) terms in turbot liver proteins (nearly 300 GO terms), while fewer GOs were found in mussel proteins (nearly 150 GO terms for FASP and S-Trap and 107 for SP3). This result may reflect the poor annotation of the genomic information in this specific group of animals. FASP was confirmed as the most consistent method for shotgun proteomic studies; however, the use of the other two methods might be important in specific experimental conditions (e.g., when samples have a very low amount of protein).
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Project description:BACKGROUND:Multiple imputation (MI) is now widely used to handle missing data in longitudinal studies. Several MI techniques have been proposed to impute incomplete longitudinal covariates, including standard fully conditional specification (FCS-Standard) and joint multivariate normal imputation (JM-MVN), which treat repeated measurements as distinct variables, and various extensions based on generalized linear mixed models. Although these MI approaches have been implemented in various software packages, there has not been a comprehensive evaluation of the relative performance of these methods in the context of longitudinal data. METHOD:Using both empirical data and a simulation study based on data from the six waves of the Longitudinal Study of Australian Children (N?=?4661), we investigated the performance of a wide range of MI methods available in standard software packages for investigating the association between child body mass index (BMI) and quality of life using both a linear regression and a linear mixed-effects model. RESULTS:In this paper, we have identified and compared 12 different MI methods for imputing missing data in longitudinal studies. Analysis of simulated data under missing at random (MAR) mechanisms showed that the generally available MI methods provided less biased estimates with better coverage for the linear regression model and around half of these methods performed well for the estimation of regression parameters for a linear mixed model with random intercept. With the observed data, we observed an inverse association between child BMI and quality of life, with available data as well as multiple imputation. CONCLUSION:Both FCS-Standard and JM-MVN performed well for the estimation of regression parameters in both analysis models. More complex methods that explicitly reflect the longitudinal structure for these analysis models may only be needed in specific circumstances such as irregularly spaced data.
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Project description:In order to meet the challenges in data evaluation and comparability between studies in multiple myeloma (MM) minimal residual disease (MRD) assessment, the goal of the current study was to provide a step-by-step evaluation of next-generation sequencing (NGS) and multicolor flow cytometry (MFC) data. Bone marrow (BM) sample pairs from 125 MM patients were analyzed by NGS and MFC MM MRD methods. Tumor load (TL) and limit of detection (LOD) and quantification (LOQ) were calculated. The best-fit MRD cut-off was chosen as 1 × 10-5, resulting in an overall 9.6% (n overall = 12 (NGS n = 2, MFC n = 10)) nonassessable cases. The overall concordance rate between NGS and MFC was 68.0% (n = 85); discordant results were found in 22.4% (11.2% (n = 14) of cases in each direction. Overall, 55.1% (n = 60/109) and 49.5% (n = 54/109) of patients with a serological response ? very good partial response (VGPR) showed BM MRD negativity by NGS and MFC, respectively. A good correlation in the TL assessed by both techniques was found (correlation coefficient = 0.8, n = 40, p < 0.001). Overall, our study shows good concordance between MM BM MRD status and TL when comparing NGS and MFC at a threshold of 10-5. However, a sufficient number of analyzed events and calculation of MRD key metrics are essential for the comparison of methods and evaluability of data at a specific MRD cut-off.
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Project description:Bottom-up proteomic strategies rely on efficient digestion of proteins into peptides for mass spectrometry analysis. In-solution and filter-based strategies are commonly used for proteomic analysis. In recent years, filter-aided sample preparation (FASP) has become the dominant filter-based method due to its ability to remove SDS prior to mass spectrometry analysis. However, the time-consuming nature of FASP protocols have led to the development of new filter-based strategies. Suspension traps (S-Traps) were recently reported as an alternative to FASP and in-solution strategies as they allow for high concentrations of SDS in a fraction of the time of a typical FASP protocol. In this study, we compare the yields from in-solution, FASP, and S-Trap based digestions of proteins extracted in SDS and urea-based lysis buffers. We performed label-free quantification to analyze the differences in the portions of the proteome identified using each method. Overall, our results show that each digestion method had a high degree of reproducibility within the method type. However, S-Traps outperformed FASP and in-solution digestions by providing the most efficient digestion with the greatest number of unique protein identifications. This is the first work to provide a direct quantitative comparison of two filter-based digestion methods and a traditional in-solution approach to provide information regarding the most efficient proteomic preparation.
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Project description:Epidemiologic studies use various biosample collection methods to study associations between human oral microbiota and health outcomes. However, the agreement between the different methods is unclear. We compared a commercially available OMNIgene ORAL kit to three alternative collection methods: Saccomanno's fixative, Scope mouthwash, and nonethanol mouthwash. Oral samples were collected from 40 individuals over 4 visits. Two samples were collected from each subject per visit: one with OMNIgene and one with an alternative method. DNA was extracted using the DSP DNA Virus Pathogen kit, and the V4 region of the 16S rRNA gene was PCR amplified and sequenced using MiSeq. Oral collection methods were compared based on alpha and beta diversity metrics and phylum- and genus-level relative abundances. All alpha diversity metrics were significantly lower for Saccomanno's fixative than for OMNIgene (P < 0.001), whereas the two mouthwashes were more similar to OMNIgene. Principal-coordinate analysis (PCoA) using the Bray-Curtis and weighted UniFrac beta diversity matrices showed large differences in the microbial compositions of samples collected with Saccomanno's compared to those with OMNIgene and the mouthwashes. Clustering by collection method was not observed in unweighted UniFrac PCoA plots, suggesting differences in relative abundances but not specific taxa detected by the collection methods. Relative abundances of most taxa were significantly different between OMNIgene and the other methods at each taxonomic level, with Saccomanno's showing the least agreement with OMNIgene. There were clear differences in oral microbial communities between the four oral collection methods, particularly for Saccomanno's fixative.IMPORTANCE We compared four different oral collection methods for studying the human oral microbiome: an OMNIgene ORAL kit, Scope mouthwash, nonethanol mouthwash, and Saccomanno's fixative. Our study shows that the type of the collection method can have a large impact on the results of an oral microbiome analysis. We recommend that one consistent oral collection method should be used for all oral microbiome comparisons. While Scope and nonethanol mouthwashes are less expensive and provide results similar to those with OMNIgene, Saccomanno's fixative may be unfavorable due to the microbial differences detected in this study. Our results will help guide the design of future oral microbiome studies.
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Project description:Two proteomic methods were recently introduced to globally map proteolytic cleavage events in biological systems, one that characterized proteolyzed proteins by differential gel migration (PROTOMAP) and the other by enzymatic tagging and enrichment of the nascent N-terminal peptides generated by proteolysis (Subtiligase). Both technologies were applied to apoptosis, and each uncovered hundreds of novel proteolytic events. An initial survey, however, revealed only minimal overlap in the two data sets. In this article, we perform an in-depth comparative analysis of the PROTOMAP and Subtiligase results that assimilates the complementary information acquired by each method. This analysis uncovered substantial agreement between the PROTOMAP and Subtiligase data sets, which in integrated form yield a highly enriched portrait of the proteome-wide impact of proteolysis in apoptosis. We discuss the respective strengths of each proteomic method and the potential for these technologies to expand the scope and sensitivity of large-scale studies of proteolysis in biological systems.
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Project description:BackgroundA number of cohort studies have collected Scope mouthwash samples by mail, which are being used for microbiota measurements. We evaluated the stability of Scope mouthwash samples at ambient temperature and determined the comparability of Scope mouthwash with saliva collection using the OMNIgene ORAL Kit.MethodsFifty-three healthy volunteers from Mayo Clinic and 50 cohort members from Bangladesh provided oral samples. One aliquot of the OMNIgene ORAL and Scope mouthwash were frozen immediately and one aliquot of the Scope mouthwash remained at ambient temperature for 4 days and was then frozen. DNA was extracted and the V4 region of the 16S rRNA gene was PCR amplified and sequenced using the HiSeq. Intraclass correlation coefficients (ICC) were calculated.ResultsThe overall stability of the Scope mouthwash samples was relatively high for alpha and beta diversity. For example, the meta-analyzed ICC for the Shannon index was 0.86 (95% confidence interval, 0.76-0.96). Similarly, the ICCs for the relative abundance of the top 25 genera were generally high. The comparability of the two sample types was relatively low when measured using ICCs, but were increased by using a Spearman correlation coefficient (SCC) to compare the rank order of individuals.ConclusionsOverall, the Scope mouthwash samples appear to be stable at ambient temperature, which suggests that oral rinse samples received by the mail can be used for microbial analyses. However, Scope mouthwash samples were distinct compared with OMNIgene ORAL samples.ImpactStudies should try to compare oral microbial metrics within one sample collection type.
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Project description:For ex vivo measurements of fracture callus stiffness in small animals, different test methods, such as torsion or bending tests, are established. Each method provides advantages and disadvantages, and it is still debated which of those is most sensitive to experimental conditions (i.e. specimen alignment, directional dependency, asymmetric behavior). The aim of this study was to experimentally compare six different testing methods regarding their robustness against experimental errors. Therefore, standardized specimens were created by selective laser sintering (SLS), mimicking size, directional behavior, and embedding variations of respective rat long bone specimens. For the latter, five different geometries were created which show shifted or tilted specimen alignments. The mechanical tests included three-point bending, four-point bending, cantilever bending, axial compression, constrained torsion, and unconstrained torsion. All three different bending tests showed the same principal behavior. They were highly dependent on the rotational direction of the maximum fracture callus expansion relative to the loading direction (creating experimental errors of more than 60%), however small angular deviations (<15°) were negligible. Differences in the experimental results between the bending tests originate in their respective location of maximal bending moment induction. Compared to four-point bending, three-point bending is easier to apply on small rat and mouse bones under realistic testing conditions and yields robust measurements, provided low variation of the callus shape among the tested specimens. Axial compressive testing was highly sensitive to embedding variations, and therefore cannot be recommended. Although it is experimentally difficult to realize, unconstrained torsion testing was found to be the most robust method, since it was independent of both rotational alignment and embedding uncertainties. Constrained torsional testing showed small errors (up to 16.8%, compared to corresponding alignment under unconstrained torsion) due to a parallel offset between the specimens' axis of gravity and the torsional axis of rotation.
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